Complex of mutualistic microbes designed to increase plant productivity

ABSTRACT

The present disclosure provides agricultural compositions and methods of using these compositions to increase plant growth, pathogen resistance and drought tolerance. The agricultural compositions disclosed herein comprise mixtures of mutualistic beneficial fungi such as  Laccaria bicolor  and  Piriformispora indica , and bacterial strains of  Pseudomonas.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority of U.S. ProvisionalApplication No. 62/246,394, filed Oct. 26, 2015, and U.S. ProvisionalApplication No. 62/294,048, filed Feb. 11, 2016, the entire contents ofwhich are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Prime Contract No.DE-AC05-000R22725 awarded by the U.S. Department of Energy. Thegovernment has certain rights in the invention.

BACKGROUND ART

The majority of terrestrial plants live in association with symbioticmicroorganisms, such as fungi which facilitate their access to soilnutrients. The ectomycorrhizal (ECM) symbiosis is the most commonassociation in forest under boreal and temperate climates. Plants harbora diverse array of asymptomatic fungal foliar and root endosymbionts andhave been recovered from all examined plant taxa to date. Several nativeendophytic and mycorrhizal fungi have been isolated and characterized aspromoter of plant host growth and productivity. Concomitantly nativebacterial isolates having a plant growth promoting effect and enhancingthe fungal-plant beneficial interaction have been characterized as well.

Populus is a dominant perennial component of temperate forests havingthe broadest geographic distribution of any North American tree genus,and is a woody perennial model with high value in pulp, paper andbiofuel industries. Populus is cultivated worldwide for pulp and paper,veneer, packing material, engineered wood products (e.g., orientedstrand board), lumber, and has recently emerged as the preeminentfast-growing woody crop for bioenergy research. Populus can be grown oneconomically marginal agricultural land thereby minimizing thecompetition between food and fuel production. Moreover, Populus is knownto associate with a wide variety of root symbiotic microbes. Populus isalso one of the few plants known to be colonized by both endo- andectomycorrhizal fungi, making it a unique model system for the study ofinteractions between plants and microorganisms.

Corn (Zea mays, also known as maize) is the most widely grown grain cropthroughout the Americas, and a food crop model for bioethanol productionin the United States.

The symbiotic fungus Laccaria bicolor is a member of Hydnangiaceae(Basidiomycota, Agaricomycotina, Agaricomycetes, Agaricomycetidae,Agaricales), a large family of ectomycorrhizal and saprotrophicbasidiomycetes. Piriformospora indica (Hymenomycetes, Basidiomycota) isa cultivable endophyte that colonizes roots.

The symbiotic fungus Hebeloma is a member of Hymenogastraceae(Basidiomycota, Agaricomycotina, Agaricomycetes, Agaricales,Hymenogastraceae), a large family of ectomycorrhizal and saprotrophicbasidiomycetes with a wide range of tree-hosts, and can be found in mostwoodland ecosystems worldwide. The symbiotic fungus Cenoccocum is anascomycetous fungus placed into the Dothideomycetes, where it representsthe only known ectomycorrhizal species within this large andecologically diverse class of Ascomycota. It is one of the most commonand globally abundant genera of ectomycorrhizal fungi, forming blackectomycorrhizas with darkly pigmented hyphae. It has a broad host- andhabitat range.

Microbial pathogens, like Atractiella (Atr) and Neonectria, aredetrimental to plant growth. Effective and eco-friendly ways are neededto combat pathogenic microorganisms. Currently the chemical compounds tocombat pathogens are also damaging to the environment and possibly humanhealth. The current invention offers a solution with a composition madeup of benign and natural microorganisms.

Another important aspect of plant growth is the ability to adapt to aridor drought conditions, which is a trait known as droughttolerance/resistance. Arid conditions can lower the yield of many crops,causing great financial losses. Therefore, methods to increase droughttolerance/resistance in plants are needed. The symbiotic microorganismcompositions disclosed herein help plants tolerate dry conditionsbetter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Root colonization rates of different L. bicolor strains ondifferent Poplar genotypes. Different Poplar genotypes (particularlypoplar genotype 52 225 (P. deltoides), poplar genotype D124 (hybridTXD), poplar genotype ILL 101 (hybrid TXD) and poplar genotype 93 968(P. trichocarpa) were treated with various L. bicolor strains(particularly Lb 445.79, Lb 559.96, Lb 594.96, Lb 560.89, Lb 669.97, Lb561.97, and Lb S238N) and root colonization percent was measured. Ineach group of bars in the Figure, from left to right, are poplargenotype 52 225 (P. deltoides), poplar genotype D124 (hybrid TXD),poplar genotype ILL 101 (hybrid TXD) and poplar genotype 93 968 (P.trichocarpa). Each group of poplar genotypes were treated with L.bicolor strains, from left to right, Lb 445.79, Lb 559.96, Lb 594.96, Lb560.89, Lb 669.97, Lb 561.97, and Lb S238N. Lb S238N gave superior rootcolonization across all poplar genotypes tested.

FIG. 2. Pseudomonas strain GM41 enhances plant growth when given withdifferent L. bicolor strains. Populus trichocarpa was incubated eitherwith different L. bicolor strains alone (particularly Lb 445.79, Lb559.96, Lb 594.96, Lb 560.89, Lb 669.97, Lb 561.97, or Lb S238N) (left,labeled a-g) or in combination with Pseudomonas fluorescens strain GM41(right, labeled a-g). Addition of GM41 significantly enhances theabove-ground fresh biomass of P. trichocarpa.

FIG. 3. Biomass production of corn plants treated with pathogenic orbeneficial fungi. Bars from left to right represent biomasses of cornplants treated with Atractiella (Atr), Atractiella and P. indica (Pir),Atractiella and L. bicolor (Lac), Atractiella and P. indica (Pir) and L.bicolor (Lac), Control, and finally Neonectria. Control means not fungalinoculation occurred; Atractiella (Atr) and Neonectria are pathogenic tocorn, while P. indica (Pir) and L. bicolor (Lac) significantly increasetotal plant biomass (roots and shoots) they also appear to rescue plantsfrom colonization by a pathogenic fungus.

FIG. 4. Metabolites affected by beneficial fungi Lac and Pir. Potentialcausal mechanisms for results found in FIG. 2 are identified in thistable. When both Lac and Pir are present in combination with thepathogen metabolites responsible for increase pest resistance areup-regulated compared to control treatments. Interestingly theseinhibitory metabolites are also involved in pathways leading toherbivore resistance and/or deterrence.

FIG. 5. Corn growth in growth chamber, responding to inoculation withpathogenic or mutualistic fungi. Pathogenic fungal inoculations on lefthand side of picture; mutualistic inoculation treatments on right handside of picture.

FIG. 6. Poplar growth in response to commensalistic, mutualistic, andpathogenic (antagonistic) fungi under greenhouse conditions. Plantstreated with commensalistic, mutualistic or pathogenic fungi are grownin the green house. Plants are randomly arranged.

FIGS. 7A-7C. Effects of microbial inoculants in Populus grown over 3months under well watered- and water-stressed (−1 MPa) conditions. P:Populus control plant; B: helper bacteria (Pseudomonas), F: fungusLaccaria. Experiments have been performed using 4 replicates ofinoculated non-inoculated Populus cuttings (ANOVA p<0.05); a aresignificantly different, b are significantly different. FIG. 7A showsplant heights, FIG. 7B shows above-ground fresh biomass, and FIG. 7Cshows total leaf area of the Populus plants under different treatments.

FIG. 8. Germination frequency of corn kernel in greenhouse. In eachgroup of bars in the figure are observed percent germination frequency(left) and expected germination frequency (right) of corn kernels. C:control (no inoculation); LP: inoculation with Laccaria bicolor andPiriformispora indica; P: inoculation with Piriformispora indica; L:inoculation with Laccaria bicolor. * Significantly different ANOVAP<0.05.

FIG. 9. Aboveground biomass of corn grown after 6-weeks growth ingreenhouse. The bars in the Figure, from left to right, are C: controlno inoculation; P: inoculation with Piriformispora indica; L:inoculation with Laccaria bicolor ; LP: inoculation with Laccariabicolor and Piriformispora indica.

FIG. 10. Germination frequency of corn kernel in field. In each group ofbars in the figure are observed percent germination frequency (left) andexpected germination frequency (right) of corn kernels. SAFE—No N:inoculation with Laccaria bicolor, Piriformispora indica and no nitrogenfertilization; SAFE—½N: inoculation with Laccaria bicolor,Piriformispora indica and nitrogen fertilization during half of thegrowing season; SAFE—Full N: inoculation with Laccaria bicolor,Piriformispora indica and nitrogen fertilization during the full growingseason; Buffer zones: no inoculation and no nitrogen fertilization.

FIG. 11. Average content of phosphorus, ammonia nitrogen and nitratenitrogen in the soil (mg/kg) inoculated with SAFE microbial mix with nonitrogen fertilization. Phosphorus (P) (first bar in each group),ammonia nitrogen (NH4—N) (second bar in each group) and nitrate nitrogen(NO₃—N) (third bar in each group) were measured 1: Before planting(June); 2: During the growing season (Aug); and 3: At harvest (Sep). Theconcentrations are measured in mg/kg.

FIG. 12. Average height of corn plant (cm) at the end of the growingseason in field. No SAFE No N: no inoculation with SAFE microbial mix(L. bicolor and P. indica) and no nitrogen-fertilization (the first barin the figure); SAFE No N: inoculation with SAFE microbial mix (L.bicolor and P. indica) and no nitrogen-fertilization (the second bar inthe figure); No SAFE +N: no inoculation with SAFE microbial mix (L.bicolor and P. indica) and nitrogen-fertilization (the third bar in thefigure); SAFE +N: inoculation with SAFE microbial mix (L. bicolor and P.indica) and nitrogen-fertilization (the fourth bar in the figure).

FIG. 13A-13B. Photos of the corn experimental field deployment. A:Planting in June B: At harvest in September of the same year.

FIG. 14A-14I: Effect of Pseudomonas fluorescens GM41 on the growth ofLaccaria bicolor, Cenococcum spp. PMI1 and Hebeloma spp. PMI1 in invitro co-cultures. P. fluorescens GM41 co-culture has a positive effecton the growth of the three fungi included in the PMB microbial mixture.FIG. 14A: Photo of in vitro cultured L. bicolor S238N; FIG. 14B:Diameter of L. bicolor S238N colonies cultured with (top curve) orwithout (bottom curve) GM41; FIG. 14C: Fungal dry weight of L. bicolorS238N cultured with (top curve) or without GM41 (bottom curve); FIG.14D: Photo of in vitro cultured Cenococcum PMI1;FIG. 14E: Diameter ofCenococcum PMI1 colonies cultured with (top curve) or without (bottomcurve) GM41; FIG. 14F: Fungal dry weight of Cenococcum PMI1 culturedwith (top curve) or without (bottom curve) GM41; FIG. 14G: Photo of invitro cultured Hebeloma spp. PMI1; FIG. 14H: Diameter of Hebeloma spp.PMI1 colonies cultured with (top curve) or without (bottom curve) GM41;FIG. 14L: Fungal dry weight of Hebeloma spp. PMI1 cultured with (topcurve) or without (bottom curve) GM41.

FIG. 15. Effects of microbial inoculants in Populus tremula X P. albagrown over 3 months in greenhouse. In each group of bars in the figureare, from left to right, aboveground fresh biomass, stem fresh biomassand leaf fresh biomass. P: Populus control plant; B: helper bacteria(Pseudomonas fluorescens GM41), F: fungal mixture of Laccaria bicolor,Cenococcum spp. PMI1 and Hebeloma spp. PMI1. Experiments were performedusing 4 replicates of inoculated or non-inoculated Populus cuttings; *significantly different ANOVA p<0.05

FIG. 16. Effects of microbial inoculants on the total leaf area inPopulus tremula X P. alba grown over 3 months in greenhouse. P: Populuscontrol plant; B: helper bacteria (Pseudomonas fluorescens GM41), F:fungal mixture of Laccaria bicolor, Cenococcum spp. PMI1 and Hebelomaspp. PMI1. Experiments were performed using 4 replicates of inoculatedor non-inoculated Populus cuttings; * significantly different ANOVAp<0.05

FIG. 17. Effects of SAFE inoculant on the aboveground biomass of Populustrichocharpa and P. deltoides under well-watered- and drought (−0.5 to−1 MPa) conditions over 3 months in greenhouse. In each group of bars,from left to right, are Pt: P. trichocarpa; Pd: P. deltoides; Pt +SAFE:mixture of P. trichocarpa and Pseudomonas fluorescens GM41, Laccariabicolor, Cenococcum spp. PMI1 and Hebeloma spp. PMI1. Pd +SAFE: mixtureof P. deltoides and Pseudomonas fluorescens GM41, Laccaria bicolor,Cenococcum spp. PMI1 and Hebeloma spp. PMI1. The group of bars on theleft represent plants grown under well-watered conditions, and the groupof bars on the right represent plants grown under drought conditions.Experiments were performed using 4 replicates of inoculated ornon-inoculated Populus cuttings; *, ** significantly different ANOVAp<0.05.

FIG. 18. Effects of SAFE inoculant on the total leaf area of Populustrichocharpa and P. deltoides under well-watered- and drought (−0.5 to−1 MPa) conditions over 3 months in greenhouse. In each group of barsare, from left to right, Pt: P. trichocarpa; Pd: P. deltoides; Pt +SAFE:mixture of P. trichocarpa and Pseudomonas fluorescens GM41, Laccariabicolor, Cenococcum spp. PMI1 and Hebeloma spp. PMI1. Pd +SAFE: mixtureof P. deltoides and Pseudomonas fluorescens GM41, Laccaria bicolor,Cenococcum spp. PMI1 and Hebeloma spp. PMI1. The group of bars on theleft represent plants grown under well-watered conditions, and the groupof bars on the right represent plants grown under drought conditions.Experiments were performed using 4 replicates of inoculated ornon-inoculated Populus cuttings; *, ** significantly different ANOVAp<0.05.

FIG. 19. Effects of SAFE inoculant on the fresh root biomass of Populustrichocharpa and P. deltoides under well-watered- and drought (−0.5 to−1MPa) conditions over 3 months in greenhouse. In each group of barsare, from left to right, Pt: P. trichocarpa; Pd: P. deltoides; Pt +SAFE:mixture of P. trichocarpa and Pseudomonas fluorescens GM41, Laccariabicolor, Cenococcum spp. PMI1 and Hebeloma spp. PMI1. Pd +SAFE: mixtureof P. deltoides and Pseudomonas fluorescens GM41, Laccaria bicolor,Cenococcum spp. PMI1 and Hebeloma spp. PMI1. The group of bars on theleft represent plants grown under well-watered conditions, and the groupof bars on the right represent plants grown under drought conditions.Experiments were performed using 4 replicates of inoculated ornon-inoculated Populus cuttings; *, ** significantly different ANOVAp<0.05.

FIG. 20A-20B. Effects of SAFE inoculant on the Superoxide radicalcontent of leaves (FIG. 20A) and roots (FIG. 20B) in Populustrichocharpa and P. deltoides under well-watered- and drought (−0.5 to−1 MPa) conditions over 3 months in greenhouse. W: well-wateredcondition; D: drought condition. SAFE: mixture of Pseudomonasfluorescens GM41, Laccaria bicolor, Cenococcum spp. PMI1 and Hebelomaspp. PMI1. Experiments were performed using 4 replicates of inoculatedor non-inoculated Populus cuttings; *, ** significantly different ANOVAp<0.05.

FIG. 21. Effects of SAFE inoculant on the Superoxide Oxide Dismutase(SOD) activity of leaves in Populus trichocharpa and P. deltoides underwell-watered- and drought (−0.5 to −1 MPa) conditions over 3 months ingreenhouse. W: well-watered condition; D: drought condition. SAFE:mixture of Pseudomonas fluorescens GM41, Laccaria bicolor, Cenococcumspp. PMI1 and Hebeloma spp. PMI1. Experiments were performed using 4replicates of inoculated or non-inoculated Populus cuttings; *, **significantly different ANOVA p<0.05.

FIG. 22. Effects of SAFE inoculant on the Peroxidase (POD) activity ofleaves in Populus trichocharpa and P. deltoides under well-watered- anddrought (−0.5 to −1 MPa) conditions over 3 months in greenhouse. W:well-watered condition; D: drought condition. SAFE: mixture ofPseudomonas fluorescens GM41, Laccaria bicolor, Cenococcum spp. PMI1 andHebeloma spp. PMI1. Experiments were performed using 4 replicates ofinoculated or non-inoculated Populus cuttings; *, ** significantlydifferent ANOVA p<0.05.

FIG. 23. Effects of SAFE inoculant on the proline content of leaves inPopulus trichocharpa and P. deltoides under well-watered- and drought(−0.5 to −1 MPa) conditions over 3 months in greenhouse. W: well-wateredcondition; D: drought condition. SAFE: mixture of Pseudomonasfluorescens GM41, Laccaria bicolor, Cenococcum spp. PMI1 and Hebelomaspp. PMI1. Experiments were performed using 4 replicates of inoculatedor non-inoculated Populus cuttings; *, ** significantly different ANOVAp<0.05.

FIG. 24A-24B. Field deployment with or without SAFE inoculation ofGreenWood Resources, Inc. commercial genotypes of Populus in Westport,Ore. A: beginning of the growing season in April; B: end of the growingseason in October of the same year.

FIG. 25A-25B. Plant size (FIG. 25A) and survival (FIG. 25B) after onemonth of growth in field with or without SAFE inoculation. A total of 60inoculated and 60 non-inoculated plants have been used for statisticalanalyses. FIG. 25A represents plant size after one month of growth infield with (left bar) and without (right bar) SAFE microbial mixture.FIG. 25B represents survival of the plants to natural pathogens afterone month of growth in field with (left bar) and without (right bar)SAFE microbial mixture.

FIG. 26. Plant height (in inch) after four months of growth in fieldwith or without SAFE inoculation. The first bar (solid) in each grouprepresents a Populus plant inoculated with SAFE mixture (S) and thesecond bar (empty) in each group represents a Populus plant that is notinoculated with SAFE mixture (NS). A total of 60 inoculated and 60non-inoculated plants have been used for statistical analyses.Experiments were performed using 12 replicates of inoculated ornon-inoculated Populus cuttings of 5 commercial genotypes from GreenWoodResources, Inc.; *, ** significantly different ANOVA p<0.05.

FIG. 27. Stem diameter (in mm) of Populus plants after four months ofgrowth in field with or without SAFE inoculation. The first bar in eachgroup represents a Populus plant inoculated with SAFE mixture (S), andthe second bar in each group represents a Populus plant that is notinoculated with SAFE mixture (NS). A total of 60 inoculated and 60non-inoculated plants were used for statistical analyses. Experimentswere performed using 12 replicates of inoculated or non-inoculatedPopulus cuttings of 5 commercial genotypes from GreenWood Resources,Inc.; *, ** significantly different ANOVA p<0.05.

FIG. 28. Root colonization rate by in situ soil microbes (and SAFEmicrobes when inoculated). The first bar in each group represents aPopulus plant inoculated with SAFE mixture (S) and the second bar ineach group represents a Populus plant that is not inoculated with SAFEmixture (NS). A total of 60 inoculated and 60 non-inoculated plants wereused for statistical analyses. Experiments were performed using 12replicates of inoculated or non-inoculated Populus cuttings of 5commercial genotypes from GreenWood Resources, Inc.; *, ** significantlydifferent ANOVA p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

Both fungal and bacterial isolates have a wide geographic and plant hostspecies distribution. So far only a few studies have examined the roleof constructed microbial communities for plants. However, these studieslacked data on constructed microbial communities on woody hosts. Thesestudies have also failed to identify mechanisms that are critical toproduce stable, scalable, mutualistic outcomes. Most research to-date onutilization of mutualistic symbiotum to increase crop fitness has a poorfield-trial record. One possible explanation for the failure in thefield is that most past research has focused on a single species of amutualistic organism in the field and it is possible that a singlemicroorganism may not be able to compete with the residentmicroorganisms in the heterogeneous environment of the soil. Researchinto utilization of complex, constructed microbial communities toincrease not only plant host yields but also the efficacy of thisapproach in a diversity of hosts and geographic contexts, is almostnon-existent.

The present inventors have investigated the impact of a constructedmultiple-microbial member community (hereinafter “proprietary microbialblend” or “PMB” or “PMB mixture” or “SAFE mixture”) on poplar and corngrowth and found significantly higher biomass yields at the greenhousescale relative to controls not exposed to the constructed microbialcommunity. PMB is a microbial composition including at least twobeneficial fungi, or a mixture of one or more beneficial fungi withbacteria that have been categorized as Mycorrhiza Helper Bacteria (MHB),such as strains of Pseudomonas fluorescens. The presence of more thanone beneficial microorganism helps establish the beneficial endo- orectomycorrhizal (ECM) symbiosis when the beneficial microorganismscooperate, and also provides redundancy in many different contexts, forexample, where a single beneficial microorganism may be competed out byresident microorganisms in the soil.

PMB can be formulated as liquid or solid formulations (such asgranulated or powdered pellets, or prills), as well as in slow orcontrolled release formulations. In solid formulations, PMBs can includebinding agents such as pullulan, paraffin, pitch or calcium nitrate.

There are many pathogenic microorganisms in the soil, and when a plantis infected with a pathogen, the growth of the plant is blunted.Pathogens cause a decline in plant biomass production and crop yield.The present inventors discovered that PMB that contains at least twobeneficial fungi, such as L. bicolor and P. indica, can preventpathogen-induced growth suppression (FIG. 3). Some embodiments of thePMB further contain at least one additional other beneficial fungalstrain, such as Hebeloma spp. and Cenoccocum spp. Combination ofbeneficial fungi in PMB provides redundancy to the microbial blend andmakes it useful in different soil conditions and with different plantvarieties.

The inventors have also discovered that PMB induces beneficial changesin host plant metabolite profile, which, without being bound in any onetheory, may explain the increased pest and pathogen resistance by thehost plant (FIG. 4).

The inventors have also reported several Pseudomonas fluorescensstrains, including isolates GM21, GM25, GM30, GM48, GM49, GM50, GM55,GM60, GM67, GM74, GM78, GM79, GM80, GM84, GM102, GM18 and GM41, thatdisplay mycorrhizal helper effect when used with beneficial fungi. Amongall the strains, GM41 showed the most beneficial effect. Pseudomonasfluorescens GM41 strain was deposited at the American Type CultureCollection (ATCC, 10801 University Boulevard, Manassas, Va. 20110 USA)on Jan. 20, 2016. The deposit was made under the terms of the BudapestTreaty. GM41 has been assigned the Accession number PTA-122788.

The inventors have observed that, in combination with a mycorrhizalfungal strain such as L. bicolor, GM41 enhances the beneficial effectthe fungal strain has on plant, further increasing plant yield andstress tolerance (FIG. 7A-7D).

Therefore, this disclosure provides compositions comprising PMB andmethods of using PMB for improving resistance to pests, increasing plantyields (biomass and fruit/grain), and improving resistance/tolerance toabiotic stresses such as salt and drought.

In one aspect, this disclosure provides compositions useful forpromoting plant growth and improving plant resistance to pests and tostress conditions.

To prepare the compositions of this disclosure, fungal inocula can beprepared in 1 L of sterile soil or 1 L of millet. After total fungalgrowth (i.e., growth to saturation), a fungal inoculum of 1 L isobtained. This inoculum is mixed with a plant soil to obtain a finalsoil used to grow plants. The term “volume to volume” refers to a ratioof the volume of an inoculum to the volume of final soil; e.g., 1 Linoculum soil with 9 L of plant soil makes 10% volume to volume. Forfield applications, 5 to 20% volume to volume (i.e., volume of inoculumsoil vs. volume of final soil) is generally used, which is equivalent to1 L to 4 L of fungal inoculum/m² of final soil. The final concentrationrange of fungi in the final soil is generally 5 to 20 fungal cells/g offinal soil.

In another embodiment, fungal inocula can be prepared in 1 L of culturemedia (e.g., potato dextrose broth). After total fungal growth (i.e.,growth to saturation), liquid culture are mixed with sterile water tountil a concentration of 50 cells (per fungal species)/ml. This finalfungal solution is mixed along with an alginic acic/alginate solution(10 grams/Liter). This mixture can either 1) be poured into a 100g/L-CaCl₂ solution with a burette and dropped as small pellets (−5 mm)to encapsulate the fungal solution in alginate beads; or 2) be poured ongranulates or soil beads then transferred to a 100 g/L-CaCl₂ solution tocoat the fungal solution on the granulates or soil beads. The finalconcentration range of fungi in the final soil is generally 5 to 20fungal cells/g of final soil.

In another embodiment, 5 ml of a liquid bacterial culture at aconcentration of 1×10²−1.6×10⁶ CFU/ml can be applied per L of final soilor culture media containing fungi as described above. For example, ifthe total soil volume is 10 L (1 L fungal inoculum+9 L of final soil),50 ml of a liquid bacterial culture can be added to the soil.

In yet another embodiment, bacteria and fungi can be co-cultured in thesame inoculum soil.

In one embodiment, a composition includes a mixture of a L. bicolorstrain and a P. indica strain. In some embodiments, the compositioncomprises a mixture of a L. bicolor strain and a P. indica strain atabout 100 total fungal cells per gram of the composition, or about 100fungal cells per strain per gram of the composition. The term “about100” refers to any number falling within the range of 85-115, i.e.,including 85, 90, 95, 100, 105, 110 and 115. In one specific embodiment,the L. bicolor is of the strain S238N.

In another embodiment, a composition includes a mixture of a L. bicolorstrain and a P. indica strain, and further includes a strain ofPseudomonas fluorescens. In specific embodiments, the compositionincludes between 1×10² CFU and 1.6×10⁶ CFU bacterial cells permilliliter of the composition. In a particular embodiment, thecomposition includes a mixture of a L. bicolor strain and a P. indicastrain, and a strain of Pseudomonas fluorescens at 5×10² CFU/mL of thecomposition. In some embodiments, the strain of Pseudomonas fluorescensis selected from one of the following isolates: GM21, GM25, GM30, GM48,GM49, GM50, GM55, GM60, GM67, GM74, GM78, GM79, GM80, GM84, GM102, GM18,and pGM41. In a specific embodiment, the strain of Pseudomonasfluorescens is GM41.

In still another embodiment, a composition includes a mixture of L.bicolor strain and a strain of Pseudomonas fluorescens. In specificembodiments, the composition includes a mixture of a L. bicolor strainat about 100 fungal cells per gram of the composition and a liquidbacterial inoculum between 1×10² CFU/mL and 1.6×10⁶ CFU/mL. In aspecific embodiment, the composition includes 5×10² CFU/mL of aPseudomonas fluorescens strain. In a specific embodiment, the strain ofL. bicolor is S238N and the strain of Pseudomonas fluorescens isselected from one of the following isolates: GM21, GM25, GM30, GM48,GM49, GM50, GM55, GM60, GM67, GM74, GM78, GM79, GM80, GM84, GM102, GM18and GM41. In a specific embodiment, the strain of Pseudomonasfluorescens is GM41.

In another embodiment, a composition includes a mixture of a L. bicolorstrain, a strain of Pseudomonas fluorescens and a strain of Cenoccocumspp. In specific embodiments, the composition includes a mixture of a L.bicolor strain at about 100 fungal cells per gram of the composition, aCenoccocum spp. strain at about 100 fungal cells per gram of thecomposition and a liquid bacterial inoculum between 1×10² CFU/mL and1.6×10⁶ CFU/mL. In a specific embodiment, the composition includes 5×10²CFU/mL of a Pseudomonas fluorescens strain. In a specific embodiment,the strain of L. bicolor is S238N and the strain of Pseudomonasfluorescens is selected from one of the following isolates: GM21, GM25,GM30, GM48, GM49, GM50, GM55, GM60, GM67, GM74, GM78, GM79, GM80, GM84,GM102, GM18 and GM41. In a specific embodiment, the strain ofPseudomonas fluorescens is GM41. In a specific embodiment, the strain ofCenoccocum spp. is PMI1.

In even yet another embodiment, a composition includes a mixture of a L.bicolor strain, a strain of Pseudomonas fluorescens, a strain ofCenoccocum spp. and a strain of Hebeloma spp. In specific embodiments,the composition includes a mixture of a L. bicolor strain at about 100fungal cells per gram of the composition, a Hebeloma spp. strain atabout 100 fungal cells per gram of the composition, a Cenoccocum spp.strain at about 100 fungal cells per gram of the composition and aliquid bacterial inoculum between 1×10² CFU/mL and 1.6×10⁶ CFU/mL. In aspecific embodiment, the composition includes 5×10² CFU/mL of aPseudomonas fluorescens strain. In a specific embodiment, the strain ofL. bicolor is S238N and the strain of Pseudomonas fluorescens isselected from one of the following isolates: GM21, GM25, GM30, GM48,GM49, GM50, GM55, GM60, GM67, GM74, GM78, GM79, GM80, GM84, GM102, GM18and GM41. In a specific embodiment, the strain of Pseudomonasfluorescens is GM41. In a specific embodiment, the strain of Hebelomaspp. is PMI1. In a specific embodiment, the strain of Cenoccocum spp. isPMI1.

In another embodiment, a composition includes a mixture of a L. bicolorstrain, a strain of Pseudomonas fluorescens and a strain of Hebelomaspp. In specific embodiments, the composition includes a mixture of a L.bicolor strain at about 100 fungal cells per gram of the composition, aHebeloma spp. strain at about 100 fungal cells per gram of thecomposition and a liquid bacterial inoculum between 1×10² CFU/mL and1.6×10⁶ CFU/mL. In a specific embodiment, the composition includes 5×10²CFU/mL of a Pseudomonas fluorescens strain. In a specific embodiment,the strain of L. bicolor is S238N and the strain of Pseudomonasfluorescens is selected from one of the following isolates: GM21, GM25,GM30, GM48, GM49, GM50, GM55, GM60, GM67, GM74, GM78, GM79, GM80, GM84,GM102, GM18 and GM41. In a specific embodiment, the strain ofPseudomonas fluorescens is GM41. In a specific embodiment, the strain ofHebeloma spp. is PMI1.

In yet another embodiment, a composition includes a mixture of a P.indica strain and a strain of Pseudomonas fluorescens. In someembodiments, the composition includes a mixture of a P. indica strain atabout 100 fungal cells per gram of the composition and a liquid inoculumof a strain of Pseudomonas fluorescens at between 1×10² CFU/mL and1.6×10⁶ CFU/mL. In a specific embodiment, the composition contains 5×10²CFU/mL of a strain of Pseudomonas fluorescens. In specific embodiments,the strain of Pseudomonas fluorescens is selected from one of thefollowing isolates GM21, GM25, GM30, GM48, GM49, GM50, GM55, GM60, GM67,GM74, GM78, GM79, GM80, GM84, GM102, GM18, and GM41. In a specificembodiment, the strain of Pseudomonas fluorescens is GM41.

The compositions disclosed herein can include, in addition to thedesirable microorganisms, other components suitable for plant growth,including phosphorus and certain trace elements such as copper, iron,manganese, zinc, cobalt, molybdenum, and boron, as oxides or saltscontaining the elements in the cationic form.

The compositions disclosed herein, if necessary, may also containadditional components suitable for agriculture or fertilizer use, suchas a water-soluble material. Suitable water-soluble materials arestarch, dextrin, gum arabic, gelatin, casein, glue, methylcellulose,carboxymethylcellulose, hydroxyalkylcellulose, alginic acid,polyvinylalcohol, polyacrylic acid, polyacrylamid, and a modified formthereof substituted partially by hydrophobic radicals in place ofhydrophilic radicals. Among these water-soluble materials, starch,dextrin, gum arabic, gelatin, casein, glue, methylcellulose,carboxymethylcellulose and hydroxyalkylcellulose are preferred. Theadditional incorporation of said water-soluble material is optional andthe amount to be added is not limited, but is usually up to 50 parts byweight for 100 parts by weight of final composition.

The compositions disclosed herein, if necessary, may also contain abinding agent selected from the group consisting of pullulan, paraffin,pitch and calcium nitrate. The present molded composition may bemanufactured by any shaping method; the customary granulation technique,customary compression molding technique or customary extrusion moldingtechnique may be conveniently used.

The compositions disclosed herein can be in solid form, and can also bein liquid form; and can include, in addition to the desirablemicroorganisms, other components suitable for plant growth, includingphosphorus and certain trace elements such as copper, iron, manganese,zinc, cobalt, molybdenum, and boron, as oxides or salts containing theelements in the cationic form in an agriculturally acceptable liquidcarrier such as water.

According to the present methods, the soil in a field can be treatedwith a composition disclosed herein at 1 L/m² to 10 L/m². One specificembodiment treats the soil in the field with 5 L/m² of a compositionmixture (i.e. 5 liters of PMB per 1 m² of soil). In some embodiments, acomposition containing a fungal inoculum of about 100 fungal cells/gramis applied to a final soil at a 5 to 20% volume to volume ratio (i.e.,volume of inoculum soil vs. volume of final soil) to achieve about 5 to20 fungal cells/gram of the final soil. In other embodiments, acomposition additionally contains a bacterial strain at a concentration1×10² CFU/mL −1.6×10⁶ CFU/mL is applied to a final soil at a 5 to 20%volume to volume dilution.

In another aspect, the disclosure provides methods of treating plantswith a microbial composition disclosed herein. The compositionsdisclosed herein can be applied to a wide array of crops (bioenergy,forage, and food), worldwide and are believed to increase nutrientavailability in soils and soil quality, resulting in increased nutrientand moisture uptake by plant roots, leading to increased plant growth.

EXAMPLE 1

Root Colonization Rates of Different L. bicolor Strains on DifferentPoplar Genotypes

Materials and Methods: Root Colonization Measurements: The percentage ofmycorrhizal colonization, as described by Tagu et al. (Variation in theability to form ectomycorrhizas in the Fl progeny of an interspecificpoplar (Populus spp.) cross. Mycorrhiza 10:237-240, 2001) was determinedthree-and-a-half months after inoculation by eight observers. All theobservers observed randomly the plants within eight blocks and blockafter block. Each root system was rinsed with tap water, cut in 1-cmpieces and analyzed under a dissecting microscope. For each root system,100 apices were randomly examined and assessed as mycorrhizal ornon-mycorrhizal.

Different poplar genotypes (particularly poplar genotype 52 225 (P.deltoides), poplar genotype D124 (hybrid TXD), poplar genotype ILL 101(hybrid TXD) and poplar genotype 93 968 (P. trichocarpa) were treatedwith various L. bicolor strains (particularly Lb 445.79, Lb 559.96, Lb594.96, Lb 560.89, Lb 669.97, Lb 561.97, and Lb S238N) and rootcolonization percent was measured.

Results: Among all the L. bicolor strains, Lb S238N gave better rootcolonization than all other strains across all poplar genotypes tested(FIG. 1).

Example 2

Pseudomononas Strain GM41 Assists L. bicolor in Promoting P. trichocarpaGrowth

Materials and Methods: Various L. bicolor strains (particularly Lb445.79, Lb 559.96, Lb 594.96, Lb 560.89, Lb 669.97, Lb 561.97, and LbS238N) were applied to P. trichocarpa plants between 5-10% volume ratioalone or in combination with 5 ml/L of Pseudomonas fluorescens GM41 10²CFU/ml. The soil was treated using 5 L/m² of the mixture (i.e. 5 litersof PMB per 1 m² of soil).

Results: GM41 increased the growth of P. trichocarpa plants incombination with all L. bicolor strains tested (FIG. 2). Lb S238N strainpromoted P. trichocarpa growth the most, both alone and in combinationwith GM41 (FIG. 2).

EXAMPLE 3

Proprietary Microbial Blend (PMB) Comprising L. bicolor and P. indicaPromotes Plant Growth Even in the Presence of Pathogens

Materials and Methods: The dikaryotic mycelium of L. bicolor S238N usedin this study was grown and maintained in Petri dishes containingPachlewski agar medium P5 (Di Battista et al., 1996) and incubated at25° C. for 3 week.

Experiments have been performed using fungal inocula grown insoil/medium mixture or millet before co-culture with plants. At the timeof the co-culture with the plants, sterile potting soil has been mixedVolume to Volume with the fungal inoculum. For example, 5% Volume ofinoculum to Volume of soil mixture means that 50 ml of the fungal soilhas been mixed with 950 ml of sterile soil to grow the plant.

Results: Application of PMB, comprising a mixture of Piriformisporaindica in combination with Laccaria bicolor at 5% volume to volumeratio, equivalent to 1 L/m² of inoculum at a concentration of 5 cells/gfinal soil, resulted in increased plant growth by corn regardless ofpresent of pathogenic fungus. When pathogen was present in the absenceof the mixture, plant growth was severely stunted (see FIG. 3 and FIG.5).

EXAMPLE 4 Proprietary Microbial Blend (PMB) Treatment IncreasesBeneficial Metabolite Levels in Plants.

Materials and Methods: Metabolite profiling—Individual metabolites wereanalyzed by metabolite profiling using gas chromatography-massspectrometry (GC-MS). Briefly, 50-75 mg of finely ground fresh tissuesample were repeatedly extracted with 2.5 ml of 80% ethanol, with theextracts then combined. A 1 ml aliquot was dried in a nitrogen stream.

After dissolving the dried extracts in acetonitrile followed bytrimethylsilylation, metabolite profiling was performed by GC-MS, asdescribed elsewhere (Jung H W et al., “Priming in systemic plantimmunity”, Science 2009, 324(5923):89-91. Metabolites were identifiedbased on mass spectral fragmentation patterns of electron impactionization (70 eV) and were quantified using peak areas of acharacteristic mass-to-charge (m/z) ratio normalized to the internalstandard (sorbitol) recovered and corrected for sample weight. Some ofthe unidentified metabolites were denoted by their retention time (RT)and key m/z ratio. The metabolite data were presented as fold changes ofthe transgenic line (average of 3 independent lines with 3 replicatesfor each line) vs. the average of the controls. Statistical significancewas assessed using Student's t-test.

Results: Metabolites involved in inhibiting microbes (6-MBOA), anddeterring herbivores (HMBOA and DIMBOA) were found to be significantlyincreased in plants treated with the PMB containing L. bicolor and P.indica strains (FIG. 4). This result at least partially explains thegrowth advantage of plants treated with the PMB.

EXAMPLE 5

Proprietary Microbial Blend (PMB) Comprising L. bicolor and PseudomonasIncreases Drought Resistance in Populus Plants

Materials and Methods: Testing Drought Tolerance—Experiments have beenperformed using 4 replicates of inoculated non-inoculated Populuscuttings. Initially, the well-watered Populus plants were stressed to−0.5 MPa, at which time they were re-watered to soil capacity. After theplants have been acclimated with three dry down cycles to −0.5 MPa, theplants were further dried down to a greater stress level of −1.0 MPa.

Results: Application of L. bicolor between 5-10% volume ratio incombination with 5 ml/L of Pseudomonas fluorescens GM41 at 10² CFU/mlresulted in increased plant growth in poplar in regular condition and aswell under light drought stress. Application of L. bicolor—GM41microbial mixture enhances water stress tolerance and a fast recoveryfrom the stress with return of optimal watering conditions (FIGS.7A-7C).

EXAMPLE 6

Proprietary Microbial Blend (PMB) Comprising L. bicolor, P. indica andPseudomonas Increases the Germination Frequency of Corn Kernel inGreenhouse, But to a Lesser Extent Than P. indica Alone

Materials and Methods: Corn plants were treated with the microbialmixture comprising 50 cells/ml of Laccaria bicolor, 50 cells/ml ofPiriformispora indica and 1.4 million of CFU/ml Pseudomonas fluorescensstrain GM41. Germination frequencies of corn kernels were measured incorn plants with or without PMB inoculation.

Results: Application of L. bicolor, P. indica and P. fluorescensresulted in an increase in germination frequency of corn kernels in agreenhouse setting (FIG. 8). However, application of P. indica aloneresulted in a greater increase in germination frequency of corn kernelsthan the microbial mixture (FIG. 8).

EXAMPLE 7

Proprietary Microbial Blend (PMB) Comprising L. bicolor, P. indica andPseudomonas Did Not Result in an Increase in Aboveground Biomass in CornPlants.

Materials and Methods: Corn plants were treated with the microbialmixture comprising 50 cells/ml of Laccaria bicolor, 50 cells/ml ofPiriformispora indica and 1.4 million of CFU/ml Pseudomonas fluorescensstrain GM41.

Results: PMB microbial mix (L. bicolor and P. indica) did notsignificantly increase the aboveground biomass of corn while inoculationcompared to control (FIG. 9). Interestingly, P. indica alone resulted ina greater increase in aboveground biomass than either L. bicolor or P.indica applied alone (FIG. 9).

EXAMPLE 8

Proprietary Microbial Blend (PMB) Comprising L. bicolor, P. indica andPseudomonas Increases the Germination Frequency of Corn Kernel in Field

Materials and Methods: Corn plants were treated with the microbialmixture comprising 50 cells/ml of Laccaria bicolor, 50 cells/ml ofPiriformispora indica and 1.4 million of CFU/ml Pseudomonas fluorescensstrain GM41.

Results: Application of L. bicolor, P. indica and P. fluorescens (PMB,or SAFE microbial mixture) resulted in an increase in germinationfrequency of corn kernels in field no Nitrogen (no N), low Nitrogen (lowN) or high Nitrogen (high N) fertilization conditions (FIG. 10).

Application of L. bicolor, P. indica and P. fluorescens (PMB, or SAFEmicrobial mixture) with or without nitrogen fertilization resulted in anincrease in corn plant height as compared to non-inoculated controlsover the period of the growth season (FIG. 12).

EXAMPLE 9

Proprietary Microbial Blend (PMB) Comprising L. bicolor, P. indica andPseudomonas Increases the Nutritive Qualities of the Soil.

Materials and Methods: Phosphorus, ammonia nitrogen and nitrate nitrogenlevels were measured in a soil sample treated with PMB, but no nitrogenfertilization throughout the growing season (June through September).

Results: Application of L. bicolor, P. indica and P. fluorescensresulted in an increase in the nutritive qualities of a soil notfertilized with a nitrogen fertilizer as demonstrated by increasingphosphorus, ammonia nitrogen and nitrate nitrogen levels (FIG. 11)throughout the growing season (June through September) (FIG. 13).

Example 10

Pseudomonas fluorescens GM41 Enhances the Growth of Laccaria bicolor,Cenococum spp. PMI1 and Hebeloma spp. PMI1 in In Vitro Co-Cultures

Materials and Methods: Colony diameters (cm) and fungal dry weights (g)of the beneficial fungi L. bicolor, Cenoccocum spp. and Hebeloma spp.have been measured with or without co-culturing with P. fluorescens GM41in vitro.

Results: Co-culturing P. fluorescens GM41 with the beneficial fungi L.bicolor, Cenoccocum spp. and Hebeloma spp. positively affected thegrowth of the co-cultured fungi (FIG. 14).

Example 11

Positive Effects of Microbial Inoculants on Populus tremula X P. albaGrowth Over 3 Months in Greenhouse

Materials and Methods: Populus plants (Populus tremula X P. albaspecies) were inoculated with mutualistic beneficial fungi such asLaccaria bicolor, the natural isolate Hebeloma spp. strain PMI1, thenatural isolate of Cenoccocum spp. strain PMI, and Pseudomonasfluorescens strain GM41. The microbial mixture (PMB) used to treatPopulus comprised 50 cells/ml of Laccaria bicolor, 50 cells/ml ofHebeloma spp. strain PMI1, 50 cells/ml of Cenoccocum spp. strain PMI1and 1.4 million CFU/ml Pseudomonas fluorescens strain GM41. After 3months of growth, aboveground fresh biomass, stem fresh biomass and leaffresh biomass of the poplar were measured in plants treated with themicrobial mixture and in non-treated control plants.

Results: Soil inoculation with PMB comprising P. fluorescens GM41 and/orwith the fungal mixture of Laccaria bicolor, Cenococcum spp. PMI1 andHebeloma spp. PMI1stimulated the growth of total aboveground biomass(aboveground, stem and leaf biomass) of Populus as compared to thenon-treated control Populus plants (FIG. 15).

The leaf area is a major determinant of photosynthesis assessing thegrowth potential of the plant. Therefore, the inventors wanted tomeasure the effect of PMB on total leaf area of Populus plants. Totalsurface area of all leaves per plant (Populus tremula X P. alba) werescanned and measured using WinRhizo/Winfolia scanner and softwaresystems. The PMB microbial mixture (SAFE mixture) comprising P.fluorescens GM41 and/or with the fungal mixture of Laccaria bicolor,Cenococcum spp. PMI1 and Hebeloma spp. PMI1, was found to increase thetotal leaf surface area increasing then the potential capacity of theplant to produce more biomass as compared to their counterparts nottreated with the PMB mixture (FIG. 16).

Example 12

PMB (SAFE) Inoculant Increases the Aboveground Biomass of P.trichocharpa and P. deltoides Under Well-Watered and Drought (−0.5 MPato −1 MPa) Conditions Over 3 Months in Greenhouse.

Materials and Methods: Testing Drought Tolerance—Experiments have beenperformed using 4 replicates of inoculated non-inoculated Populuscuttings. Initially, the well-watered Populus plants were stressed to−0.5 MPa, at which time they were re-watered to soil capacity. After theplants have been acclimated with three dry down cycles to −0.5 MPa, theplants were further dried down to a greater stress level of −1.0 MPa.

Results: Inoculating different Populus strains with the PMB (aka. SAFE)mixture increased the aboveground biomass in well-watered condition aswell as under drought stress in two different other Populus species (P.trichocharpa and P. deltoides) (FIG. 17). However, the PMB mixture(SAFE) did not increase the total leaf area in Populus plants underdrought stress (FIG. 18, right bars), whereas it increased under normal(unrestricted) watering conditions (FIG. 18, left bars).

The inventors also investigated the fresh root biomass of Populus plantsunder well-watered and drought conditions. Inoculation with PMBincreased the growth of fresh biomass in well-watered condition as wellas under drought stress (FIG. 19). The PMB (SAFE) inoculant allowed theplant biomass increase observed in FIG. 16 by increasing rootdevelopment and therefore allowing a better water supply under droughtstress condition.

Using specific PCR primers, the inventors confirmed the presence, andtherefore the persistence of each microbial strain of the PMB mixture(SAFE) in soil and on plant roots after three months.

Example 13 PMB (SAFE) Mixture Protects Populus Plants Against ROS UnderWell-Watered and Drought Stress Conditions.

To measure possible effects of PMB on the stress caused by radicaloxygen species (ROS), the inventors first measured the superoxidecontent of P. trichocarpa and P. deltoides leaves (FIG. 20A) and roots(FIG. 20B) under normal (well-watered) or stress (drought) conditions asdescribed above. It was found that the superoxide radical content inPopulus leaves were higher in drought conditions as well as well wateredconditions in PMB (SAFE) mixture treated plants. On the other hand, thesuperoxide content in the roots were decreased under well-wateredcondition as well as under drought stress (FIG. 20A and FIG. 20B).

To minimize the damage from Radical Oxygen Species (ROS) plants utilizeenzymatic antioxidants such as the Super Oxide Dismutase (SOD) andperoxidase (POD). It was found that both SOD (FIG. 21) and POD activitylevels (FIG. 21) were increased in the leaves of P. trichocharpa and P.deltoides treated with the PMB mixture as compared to controls, underboth well-watered and drought (−0.5 MPa to −1 MPa) conditions over 3months in a greenhouse setting (FIG. 21 and FIG. 22).

Another plant defense mechanism against ROS damage is adjusting osmoticpressure by producing an osmoprotectant such as proline. The inventorsmeasured proline levels in Populus leaves to see the effect of PMB inplant osmoprotection. As a result, it was found that inoculation thePopulus plants with the PMB (SAFE) mixture in greenhouse increased theproline content in Populus leaves as compared to control plants,particularly under drought stress (FIG. 23).

Thus, the PMB (SAFE) inoculation facilitated protection againstdrought-induced stresses.

EXAMPLE 14 PMB (SAFE) Positively Affects Plant Size and Survival inField

Populus plants were planted in a field in Westport, Ore. (FIG. 24), withor without PMB (SAFE) inoculation.

It was observed that plants treated with the microbial mixture grew morein size (FIG. 25A) and better survival percentage (FIG. 25B) than theirnon-treated counterparts after one month in the field.

After four months in the field, the PMB (SAFE) treated plants weretaller (FIG. 26), and had a thicker stem (FIG. 27) as compared to theirnon-treated counterparts.

The current inventors have also shown in field that inoculation with thePMB (SAFE) mixture increased Populus root colonization by in situbeneficial microbes (FIG. 28). The presence and persistence of eachstrain of the SAFE microbes were assessed and confirmed on plant rootsand surrounding soil by PCR amplification using specific designed PCRprimers.

1. A composition comprising a Laccaria bicolor strain and aPiriformispora indica strain.
 2. (canceled)
 3. The composition of claim1, wherein said composition includes about 100 fungal cells per gram ofthe composition.
 4. The composition of claim 1, further comprising aPseudomonas fluorescens strain.
 5. The composition of claim 4, whereinsaid Pseudomonas fluorescens strain is the strain designated as GM41(ATCC PTA-122788).
 6. (canceled)
 7. The composition of claim 4, whereinsaid Pseudomonas fluorescens strain is present in the composition at aconcentration between 1×10² CFU/mL and 1.6×10⁶ CFU/mL.
 8. Thecomposition of claim 7, wherein said composition is a liquid formulationand further comprises components suitable for plant growth.
 9. Thecomposition of claim 7, wherein said composition is a solid formulationand further comprising components suitable for plant growth.
 10. Thecomposition of claim 9, further comprising a binding agent selected fromthe group consisting of pullulan, paraffin, pitch and calcium nitrate.11. A composition comprising: a Laccaria bicolor strain and aPseudomonas fluorescens strain.
 12. (canceled)
 13. The composition ofclaim 11, wherein said Pseudomonas fluorescens strain is the straindesignated as GM41 (ATCC PTA-122788).
 14. The composition of claim 13,wherein said Laccaria bicolor strain is present in the composition atabout 100 cells per gram, and said Pseudomonas fluorescens strain ispresent in the composition at a concentration between 1×10² CFU/mL and1.6×10⁶ CFU/mL.
 15. The composition of claim 14, wherein saidcomposition is a liquid formulation and further comprises componentssuitable for plant growth.
 16. The composition of claim 14, wherein saidcomposition is a solid formulation and further comprises componentssuitable for plant growth.
 17. The composition of claim 16, furthercomprising a binding agent selected from the group consisting ofpullulan, paraffin, pitch and calcium nitrate.
 18. The composition ofclaim 11, further comprising a Cenoccocum spp. strain, a Hebeloma spp.strain, or a combination thereof.
 19. The composition of claim 14,further comprising a Cenoccocum spp. strain, wherein said Cenoccocumspp. strain is present in the composition at about 100 cells per gram.20-22. (canceled)
 23. The composition of claim 14, further comprising aHebeloma spp. strain, wherein said Hebeloma spp. strain is present inthe composition at about 100 cells per gram.
 24. A compositioncomprising a Piriformispora indica strain and a Pseudomonas fluorescensstrain.
 25. The composition of claim 24, wherein said Pseudomonasfluorescens strain is the strain designated as GM41 (ATCC PTA-122788).26. The composition of claim 25, wherein said Piriformispora indicastrain is present in the composition at about 100 cell per gram and saidPseudomonas is present in the composition at a concentration between1×10² CFU/mL and 1.6×10⁶ CFU/mL.
 27. The composition of claim 26,wherein said composition is a liquid formulation and further comprisescomponents suitable for plant growth.
 28. The composition of claim 26,wherein said composition is a solid formulation and further comprisescomponents suitable for plant growth.
 29. The composition of claim 28,further comprising a binding agent selected from the group consisting ofpullulan, paraffin, pitch and calcium nitrate.
 30. A method comprisingtreating plants with a composition according to claim
 1. 31. A methodcomprising treating plants with a composition according to claim
 11. 32.A method comprising treating plants with a composition according toclaim
 24. 33. (canceled)